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HEMATOPOIESIS
From the Departments of Clinical Hematology and
Laboratory Hematology, University of Liège, Belgium; and the
National Fund for Scientific Research, Brussels, Belgium.
Ex vivo expansion of hematopoietic stem/progenitor cells may result
in defective engraftment. Human cord blood CD34+ progenitor
cells were synchronized and assayed for adhesion and migration onto
fibronectin (Fn) and vascular cell adhesion molecule-1 (VCAM-1) at
different stages of a first cell cycle executed ex vivo. During S phase
transit, adhesion to Fn was transiently increased while binding to
VCAM-1 was reversibly decreased, after which adhesion to both ligands
returned to baseline levels with cell cycle completion. Transmigration
across Fn and VCAM-1 decreased irreversibly during S phase progression.
The function of Treatment with cyclophosphamide and
granulocyte-colony stimulating factor (G-CSF) induces hematopoietic
stem cell (HSC) proliferation in the bone marrow (BM) and
mobilization in the peripheral blood (PB).1 Release in the
PB seems to be dependent on a cell cycle-controlled mechanism and
occurs specifically after M phase of the cell cycle.2 Similarly, only HSCs residing in G0/G1 are
present in the PB after mobilization with G-CSF.3-5
Strikingly, implantation in the BM after infusion in the PB has also
been shown to be dependent to some extent on the cell cycle status of
infused cells. Indeed, in a number of studies, it could be observed
that engraftment of human and murine HSCs was maximal during
G0/G1 phase of the cell cycle and reduced
during S and G2/M.6-8
It is generally considered that HSCs reside in specialized niches in
the BM microenvironment, in which they are sequestrated through
multiple interactions with stromal cells and extracellular matrix
molecules. Migration across the BM stroma is directed by chemokines
such as stromal derived factor-1 (SDF-1). Fibronectin (Fn) is located
in the outer lining of endothelial cells and present throughout the BM
stroma, especially in the endosteal region, where HSCs are thought to
home selectively.9,10 Conflicting results have been
reported as to whether primitive progenitor cells adhere to the
Adhesion of hematopoietic cells to Fn is not constitutive but highly
susceptible to a variety of stimuli, including
cytokines,17-20 chemokines,14 and ligation of
other adhesion molecules.21 Changes in Fn binding induced
by such stimuli are transient and dependent on affinity modulation of
VLA-4 and VLA-5 integrins. Modulation of integrin expression may also
take place and has been associated with mobilization and homing of
CD34+ cells.12,22 Fn is thus an attractive
candidate in providing modulated interactions with stem/progenitor
cells, which may be implicated in the control of their trafficking.
In addition to Fn binding, endothelial cell adhesion molecules
participate in stem/progenitor cell trafficking.23
Injection of antivascular cell adhesion molecule-1 (anti-VCAM-1)
antibody induces HSC mobilization in the peripheral blood in the same
manner as anti-VLA-4 antibody. Anti-VCAM-1 antibody also blocks
homing of progenitor cells in the bone marrow and increases their
uptake by the spleen.24,25 Furthermore, treatment of human
progenitor cells with antilymphocyte function-associated antigen-1
(anti-LFA-1) was shown to reduce short-term engraftment in
immunodeficient mice, an effect that could be related to
down-modulation of transendothelial migration through interaction of
LFA-1 with intercellular adhesion molecule-1 (ICAM-1).14
Whether long-term reconstituting HSCs express functional LFA-1 has not
yet been demonstrated.26
In the present study, using cell cycle synchronization, changes in
binding and migration on Fn, VCAM-1, and ICAM-1 of human cord blood
CD34+ cells were assessed during transit through a single
cell cycle and prior to any cell division. Transit through S phase was
associated with a reversible increase in adhesion to Fn and a
reciprocal decrease in adhesion to VCAM-1, while binding to ICAM-1 was
not significantly affected. Transmigration through all 3 ligands
decreased irreversibly during S phase transit. Our results also
demonstrate that unstimulated progenitor cells interacted with Fn via
VLA-4 while mitotically activated progenitor cells used mainly VLA-5 to
adhere and migrate onto Fn.
Cells
The murine stromal MS-5 cell line27 was plated in 25 cm2 flasks in 7 mL Synchronization cultures
Adhesion and migration assays of CD34+ cells Fn (Sigma) or 40- and 120-kDa Fn fragments (Life Technologies) were adsorbed at 4°C overnight to wells of nontissue culture-treated 24-well plates at 10 µg/cm2 in phosphate-buffered saline (PBS; Biowhittaker). Recombinant human VCAM-1 or ICAM-1 (R&D Systems) were coated at 1 µg/cm2 in tissue-culture-treated plates in PBS at 4°C overnight. Control plates were coated with 1% fraction V bovine serum albumin (BSA; Life Technologies). The coating solutions were removed by aspiration, and plates were incubated with RPMI 1640 (Biowhittaker) containing 1% BSA at 37°C for 30 minutes to block nonspecific binding sites. After 2 washes in RPMI 1640 with 25 mmol HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), adequate numbers of unmanipulated or cultured CD34+ cells were plated in coated plates in prewarmed serum-free medium without cytokines. After 1 hour of incubation at 37°C, nonadherent cells were harvested by 2 standardized washes with PBS 1% BSA. Adherent cells were recovered after a 5-minute incubation in an enzyme-free cell dissociation buffer (CDB, Life Technologies) at 37°C followed by vigorous pipetting. Percent adhesion was calculated as follows: number of adherent cells / (number of adherent cells + number of nonadherent cells).Migration assays were performed in 6.5-mm-diameter, 5-µm pore
Transwells (Costar, Cambridge, MA). Transwell filters were
coated with 10 µg/cm2 Fn, 1 µg/cm2 VCAM-1,
1 µg/cm2 ICAM-1, or 1% fraction V BSA as described
above. After 2 washes in RPMI 1640 with 25 mmol HEPES,
2 × 105 cells were plated in 100 µL serum-free medium
in the upper chamber of the transwell. The bottom compartment was
filled with 600 µL of MS-5 CM or nonconditioned medium ( Determination of LTC-IC adhesion and migration Long-term culture-initiating cell (LTC-IC) activity of various cell fractions was determined in bulk long-term cultures as previously described.29,30 Up to 3 × 104 CD34+ cells in serum-free medium without cytokines were plated in culture dishes coated with 1% BSA or 10 µg/cm2 Fn and incubated for 1 hour. Nonadherent cells were removed by gentle washing, and adherent cells were overlayed with unirradiated stromal MS-5 cells in long-term culture medium (Myelocult; Stem Cell Technologies). The LTC-IC activity of input cells was measured by plating one tenth of the original cell suspension in a BSA- or Fn-coated dish followed directly by the addition of MS-5 cells. Cultures were placed at 33°C and fed weekly by half-medium changes. After 5 weeks, cultures were trypsinized and harvested cells were replated in duplicates in 1 mL Methocult H4435 semisolid medium (Stem Cell Technologies) containing 50 ng/mL SCF, 20 ng/mL granulocyte macrophage-colony stimulating factor (GM-CSF), 20 ng/mL interleukin-3 (IL-3), 20 ng/mL IL-6, 20 ng/mL G-CSF, and 3 U/mL erythropoietin. After 2 weeks at 37°C, secondary colony-forming cells (CFCs) were scored with an inverted microscope using standard criteria. The proportion of adherent LTC-ICs was calculated as follows: number of secondary CFCs produced by adherent cells/(number of secondary CFCs produced by input cells × 10).Migration of LTC-ICs across Fn was assayed in BSA- or Fn-coated Transwells of 5-µm pore diameter. MS-5 cells were plated in 600 µL Myelocult in the lower compartment 7 days in advance to allow medium conditioning. Input cells were then placed in 100 µL serum-free medium in the upper chamber. Following migration for 3 hours, the LTC-IC content of migrating cells was determined in the lower chamber using the MS-5 cells as feeders. The total LTC-IC activity of input cells was measured by directly plating one tenth of the original cell suspension onto MS-5 cells. Bulk long-term cultures and secondary CFC enumeration were performed as described above. The proportion of migrating LTC-ICs was calculated as follows: number of secondary CFCs produced by migrating cells/(number of secondary CFCs produced by input cells × 10). Integrin blocking experiments CD34+ cells were preincubated with antihuman 4
integrin (clone P4C2; Life Technologies), antihuman 5 integrin
(clone P1D6; Life Technologies), antihuman LFA-1 (clone Hl111;
Pharmingen, San Diego, CA), or control mouse immunoglobulin G
(IgG; Pharmingen) at 1:100 dilution during 30 minutes at 4°C and were
then replated in CD34+ cell or LTC-IC adhesion and
migration assays. Inhibition of adhesion or migration was expressed
relative to adhesion or migration of IgG-treated cells, respectively.
In experiments involving LTC-IC measurements, it was preestablished
that a 30-minute incubation with anti-4 or -5 antibodies prior to
long-term cultures did not interfere with subsequent LTC-IC output
(data not shown).
Cell cycle status of LTC-ICs Briefly, 2 × 104 to 10 × 104 CD34+ cells were treated with 2 mg/mL hydroxyurea (HU; Sigma) in IMDM 1% BSA during 1 hour at 37°C as previously described.31 Control cells were incubated in medium only. After 2 washes in IMDM with 1% BSA, cells were replated in bulk LTC-IC assays as described above. The percentage of cycling LTC-ICs was estimated from the number of secondary CFCs killed by HU treatment compared with the number of secondary CFCs produced by control cells.Flow cytometric analysis of integrin expression VLA-4 expression was determined with fluorescein isothiocyanate (FITC)-conjugated antihuman4 integrin
(Coulter Immunotech, Marseille, France) or isotype-matched control IgG
(Pharmigen) for 20 minutes on ice in the dark. VLA-5 expression was
measured after labeling with unconjugated antihuman 5 integrin
(clone P1D6, Life Technologies) or control mouse IgG, followed by
staining with goat antimouse allophycocyanin (Molecular Probes, Eugene, OR). LFA-1 was analyzed by staining cells with phycoerythrin
(PE)-conjugated anti-CD11a/LFA-1 (Pharmingen) or PE-conjugated
isotypic control. Cells were washed in PBS 1% calf serum
(Biowhittaker) and fixed in PBS 1% paraformaldehyde (Sigma). Data were
acquired on a FACSort flow cytometer (Becton Dickinson [BD], San
Jose, CA). Antigen density was expressed as mean channel fluorescence
ratio (MCFR) defined as MCF of integrin expression divided by MCF of
fluorescence-matched isotypic control.
Flow cytometric analysis of cell cycle status Cells were incubated in PBS 0.6% IGEPAL CA-630 (Sigma) containing 50 µg/mL propidium iodide (PI; Sigma) and 1 mg/mL RNAse (Boehringer, Mannheim, Germany). After a 30-minute incubation on ice in the dark, cells were analyzed on a FACSort flow cytometer. The percentage of cells in G0/G1, S, and G2+M was determined using Modfit 2.0 software (BD).Statistical analysis Results are reported as mean ± SEM. Gaussian distribution of the data was assessed with Kolmogorov-Smirnov tests (SigmaStat software; SSPS, Richmond, CA). Paired Student t tests and nonparametric Wilcoxon signed rank tests were used for Gaussian and non-Gaussian distributions, respectively. All P values are 2-sided.
Synchronization of CD34+ cells and LTC-ICs In the present study, our first intent was to synchronize progenitor cells to monitor changes in adhesion and motility at various stages during a single cell cycle transit (Figure 1). The procedure adapted from Reddy et al28 effectively allowed us to follow cells in 3 distinct phases of a single cell cycle and prior to any cell division: in G0/G1 (Figure 1A), at the G1/S transition (Figure 1B), and in S phase (Figure 1D). To demonstrate that the same kinetics also applied to the primitive LTC-IC subset of the cell population, hydroxyurea LTC-IC killing assays were used (Figure 1A,B,D). The synchronization procedure was effective not only on total CD34+ cells but on primitive progenitor cells (LTC-ICs) as well. In addition, aphidicolin synchronization had no toxicity on CFCs or on LTC-ICs because CFC and LTC-IC activities of CD34+ cells harvested from cultures with or without aphidicolin were similar (Table 1).
Adhesion and migration across Fn of synchronized CD34+ cells Adhesion of CD34+ cells to Fn was determined at the following time points: when freshly isolated in the G0/G1 phase; when blocked at the G1/S transition by a 24-hour treatment by aphidicolin; after 2, 4, 6, and 8 hours following aphidicolin treatment; and, finally, 24 hours after the end of aphidicolin treatment (termed hereafter postmitotic cells). We observed a large increase in Fn binding when cells progressed from G0/G1 to the G1/S transition. During S phase, adhesion to Fn increased further and, interestingly, this process was reversible after S phase completion and progression in G2/M as well as in postmitotic cells (Figure 2A).
To explore in a more dynamic fashion the interactions of progenitor
cells with Fn, Transwell migration assays were set up with synchronized
CD34+ cells. MS-5 CM was used to provide a marrow
chemotactic gradient as previously described.30 Migration
of CD34+ cells across Fn was maximal in
G0/G1 and decreased while transiting through S
phase (Figure 2B). Contrary to what was observed in adhesion assays,
changes in migration capacity of synchronized CD34+ cells
were not reversible after S phase transit. Control experiments were
conducted to establish whether the defective migration of cycling cells
was caused by an impaired interaction with Fn or by a diminished
response to MS-5 CM. Spontaneous migration across Fn toward
nonconditioned medium ( Next, the functional state of
To exclude the possibility that the observed changes in adhesion and
migration of synchronized CD34+ cells resulted from
aphidicolin-induced alterations of the cytoskeleton as described in
adherent fibroblasts,34,35 the following control experiments were done. Freshly isolated CD34+ cells were
plated without cytokine stimulation and treated with aphidicolin for 24 hours before being washed, replated in cytokine-free medium in the
absence of aphidicolin, and sampled after 3, 6, and 24 hours
(n = 3). These conditions maintained the proportion of cells
in G0/G1 between 97.3% and 98.9% throughout
the experiment. Neither adhesion nor transmigration capacities were
affected by aphidicolin treatment: Adhesion to Fn remained stable
between 3.9% and 4.7%, and transmigration across Fn varied between
53.8% and 56.0%. The functional state of Adhesion and migration across Fn of LTC-ICs during cell cycle progression Because Fn binding may differ in primitive and committed progenitor cells,36 we specifically examined adhesion not of total CD34+ cells but of primitive progenitor cells detected as LTC-ICs (Figure 5A). On BSA-coated plates, the percentage of adherent LTC-ICs was always less than 5%. On Fn, LTC-IC adhesion rose during S phase transit and returned to baseline in postmitotic cells. Thus, as for committed CD34+ progenitor cells, S phase transit of LTC-ICs was associated with a reversible increase in adhesion to Fn. The contribution of VLA-4 and VLA-5 in mediating Fn binding of LTC-ICs was separately assessed with blocking antibodies. Adhesion of freshly isolated LTC-ICs in G0/G1 was inhibited by anti-4 but not by anti-5 integrin (Figure 5B). In contrast, in S
phase and in postmitotic LTC-ICs, 5-mediated adhesion increased
while 4-mediated Fn binding was down-modulated.
Migration of synchronized LTC-ICs toward MS-5 CM was assayed in Fn- or
BSA-coated Transwells (Figure 6A). Up to
58.7% ± 1.1% of freshly isolated LTC-ICs were able to migrate
across Fn. There was a decrease in LTC-IC migration at the
G1/S transition and prominently during S phase. This
decrease was not reversible in postmitotic cells. As for
CD34+ cells, LTC-IC migration toward MS-5 CM across BSA was
similar in all phases of the cell cycle. Dependence of LTC-IC migration toward MS-5 CM on
Adhesion and migration of synchronized CD34+ cells across VCAM-1 and ICAM-1 Adhesion and migration of synchronized CD34+ cells were measured on both VCAM-1 and ICAM-1. Contrarily to Fn binding, adhesion to VCAM-1 was down-modulated during S phase before returning to baseline levels in postmitotic cells. Adhesion to ICAM-1 was not significantly affected by cycle transit (Figure 7A). Transmigration toward MS-5 CM across both adhesion molecules decreased irreversibly during S phase transit while spontaneous migration across both ligands toward nonconditioned medium was observed in approximately 10% of the cells in all stages (Figure 7B,C).
Control experiments were carried out as described above for cell-Fn
interactions to determine potential effects of aphidicolin treatment on
binding and migration on VCAM-1 and ICAM-1, independently of cycle
progression (n = 3). In CD34+ cells kept in
G0/G1 under cytokine-free conditions in
culture, aphidicolin treatment did not induce any changes in binding or motility, either on VCAM-1 or ICAM-1. The proportion of cells binding
to VCAM-1 varied between 30.8% and 33.9% while migration across
VCAM-1 was maintained between 45.4% and 48.8%. Adhesion (range,
15.3% to 19.1%) and migration (range, 30.4% to 33.9%) on ICAM-1
were similarly unaffected by aphidicolin treatment. The functional
state of VLA-4 and LFA-1 in mediating interaction of
G0/G1 CD34+ cells with VCAM-1 and
ICAM-1, respectively, was determined with specific blocking antibodies.
Again, no changes in the activation state of either receptor were
associated with aphidicolin treatment, in both adhesion and
transmigration assays. Inhibition of VCAM-1 binding by anti- Expression of VLA-4, VLA-5, and LFA-1 by synchronized CD34+ cells We attempted to correlate the differences in integrin function observed during cell cycle transit with parallel changes in integrin expression. VLA-5 was not detected in G0/G1 CD34+ cells (Table 2 and Figure 8). VLA-5 MCFR rose at the G1/S transition (P < .05), but no further changes were seen during S phase and in postmitotic cells. As for VLA-4, MCFR was higher than that of VLA-5 in unstimulated G0/G1 CD34+ cells and roughly doubled at the G1/S transition but without reaching statistical significance. VLA-4 MCFR was statistically higher in S phase and in postmitotic cells compared with G0/G1 cells. Expression of LFA-1 was similarly up-regulated during cell cycle progression of synchronized CD34+ cells. Thus, reversible modulations in Fn and VCAM binding during S phase were not paralleled by changes in integrin expression but more likely by alterations of their functional state.
In this study, we have observed a reversible increase in Fn
binding and a reciprocal decrease in VCAM-1 binding of hematopoietic progenitor cells during transit through S phase of the cell cycle. We
used a synchronization procedure to follow cell-matrix interactions at
3 distinct stages The second important observation reported here is that mitotically
activated progenitor cells shift from a dominant A number of studies have documented a loss of engraftment potential of cycling stem/progenitor cells as compared with their quiescent counterparts.6-8 While initial seeding of transplanted cells to the BM does not appear to be influenced by cell cycle status, a recent study suggests that intramedullary homing and retention in the hematopoietic niche are specific properties of noncycling cells.40 The progressive decline of migration capacity toward a marrow chemotactic gradient that occurs during S phase across Fn, VCAM-1, and ICAM-1 further supports these observations. Moreover, if VCAM-1 is considered the prominent BM endothelial addressin,25 decreased binding to VCAM-1 in progenitor cells transiting through S phase may result in defective transendothelial migration and marrow homing. Our observations are also consistent with impaired mobilization of cycling stem/progenitor cells.2-4 Fn has been shown to be abundantly present in the BM endosteal region, where hematopoietic niches are located.9,10 Increased static binding and impaired transmigration across Fn in cycling progenitor cells may result in defective intramedullary motility. Furthermore, decreased adhesion and transmigration capacity on VCAM-1 may reduce transendothelial movements of cycle-activated cells from the BM into the PB. Because Fn is a main component of the hematopoietic niche, the
predominance of VLA-5-Fn interaction in mitotically activated cells
compared with VLA-4-Fn in resting progenitor cells may have important
implications. First, modulation of integrin function in ex vivo
cultures might be responsible for aberrant intramedullary trafficking
of expanded cells. The importance of VLA-4-Fn interaction in the BM
microenvironment has been demonstrated.11 A substitution of a dominant VLA-4-Fn interaction by a predominant VLA-5-Fn
interaction might incapacitate stem cell localization in adequate
niches during intramedullary homing and/or cause divergent lodgment of
cycling and noncycling cells and subsequent differences in survival and initiation of hematopoiesis, as suggested by Jetmore and
colleagues.40 Second, inactivation of
The authors thank Dr E. Ponthier and all the nurses and physicians of the obstetrical department of the "Cliniques St Joseph," Liège, Belgium, for providing cord blood samples. Y.B. and A.G. are Research Director and Associate Scientist, respectively, of the National Fund for Scientific Research (Brussels, Belgium).
Submitted October 3, 2001; accepted May 28 2002.
Supported by grants from the National Fund for Scientific Research, the University of Liège Center against Cancer, and the Belgian Federation against Cancer (a nonprofit organization). S.H. and O.G. were supported by Télévie fellowships.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: André Gothot, University of Liège, Laboratory Hematology, CHU Sart Tilman B35, 13, avenue de l'Hôpital, B-4000 Liège, Belgium; e-mail: agothot{at}ulg.ac.be.
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F. Ahmed, S. J. Ings, A. R. Pizzey, M. P. Blundell, A. J. Thrasher, H. T. Ye, A. Fahey, D. C. Linch, and K. L. Yong Impaired bone marrow homing of cytokine-activated CD34+ cells in the NOD/SCID model Blood, March 15, 2004; 103(6): 2079 - 2087. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
1 (very late antigen-4 [VLA-4]) integrin-binding domain
2 mmol 2-mercaptoethanol (all from
Biowhittaker). Cells were stimulated by a combination of 100 ng/mL stem cell factor (SCF), 100 ng/mL thrombopoietin (TPO; both from
Amgen, Thousand Oaks, CA), and 100 ng/mL flt3-ligand (FL; R&D Systems)
and maintained at 37°C in a 100% humidified atmosphere with 5%
CO2. After 16 hours, cells were reversibly blocked at the
G1/S transition by a 24-hour treatment with 2 µg/mL
aphidicolin (Sigma), as previously described by Reddy et
al.
40 hours); #P < .05
compared with cells blocked by aphidicolin in G1/S (0 hours). (B) Transmigration assays were carried out across Fn toward
MS-5 CM (gray bars) and control medium (white bars) or across BSA
toward MS-5 CM (black bars). *P < .05 compared with
uncultured cells.
) and
nonconditioned medium (
; n = 3). (C) Transmigration across ICAM-1
was measured toward MS-5 CM (
in G0/G1, at the
G1/S transition, and in S phase